Detalles del proyecto

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CSC - Cost-sharing contracts

Objetivo

The principal objective of this programme is to establish the feasibility of biosensor technology for the detection and quantitative analysis of pesticides, and to establish the viability of doing so in an on-stream device.

Biosensors for the detection and quantitative analysis of pesticides are being developed, based on the strong and highly specific binding of antigens (pesticides or their analogues) by antibodies, coupled with secondary electrochemical methods of signal transduction based on enzyme or whole cell reactions with monitoring by mass spectrometry (MS).

The catch system has been used which allows concentration and continuous monitoring of a model pesticide, coumarin, using affinity chromatography. The preconcentrator consists of a column which is packed with polyclonal antibody covalently coupled to an insoluble gel matrix. The coumarin molecules, in a large volume of sample passing through the preconcentrator, bind specifically to the immobilised antibodies, and are effectively extracted from solution. The bound coumarin is subsequently eluted from the preconcentrator in a small volume of solution which results in the concentration of the analyte. The sample than passes on to the detection system. By this method low concentrations of pesticides can be quantitated without the requirement of a very sensitive detection method.

A system is being developed in which pesticides are allowed to displace antigens bound to a column in proportions which can be related to the pesticide concentration. The quantity of antigen displaced from the column, and hence the pesticide concentrations, can be determined by attaching an appropriate reporter group (eg glucose oxidase) to the antigen. Electrochemical methods of enzyme assay have been used, based upon a biosensor operating in amperometric mode, employing glucose oxidase as the biological recognition component. When the glucose concentration is maintained at a constant value the amperometric signal is proportional to the quantity of enzyme within the electrode.

Mass spectrometry (MS) techniques were found to monitor coumarins and coumarin derivatives to levels as low as 2 pg per second and were reproducible confirming their use in measuring pesticide concentrations.

The improvement of conditions for fast and sensitive immunoassays includes the choice of solvents. The use of organic solvents, either pure or in solvent water mixtures, promises several advantages, as enhancement of stability, affinity, specificity and easier detection of hydrophobic analytes.

Water miscible solvents were investigated with respect to enzyme activity and stability, antibody stability, sensitivity and relative affinities. The enzymes peroxidase and laccase have been studied as marker enzymes for the anti coumarin hapten system. Laccase, which uses the same chromogenic substrates as peroxidase, but without the need for hydrogen peroxide, was shown to be inhibited by ethanol in photometric assays to a much lower extent than peroxidase. The stability of both enzymes is comparably high, with about 50% inhibition by overnight incubation in 50% ethanol. For electrochemical detection laccase has to be preferred. Luminescence was also investigated, as it is one of the most sensitive enzyme detection principles. However, laccase does not catalyse luminescence reactions, at least at the optimum conditions for the enhanced peroxidase luminol reaction.

The behaviour of anti coumarin antibodies in organic solvents was also studied. Immobilized antibodies were shown to be highly resistant even to pure ethanol. Evidence was found that methanol at low concentration improves the sensitivity in a competitive immunoassay. It remains to be seen if such solvent water mixtures also lead to improved displacement immunoassays.

With respect to elution of antibody bound analytes it is planned to study solvent buffer mixtures, which allow either direct measurement of the eluted analyte with a biomonitor or, for competitive flow immunoassays, the simultaneous enzyme reaction. In the latter case shorter assay times can be achieved by reduction of steps.

Electrodes made up of cyanobacteria have been used as a biosensor for immediate detection of acute toxic effects of pollutants. Monocellular cyanobacteria like Synechococcus and Microcystis were found to be more sensitive than chain linked cyanobacteria like Nostoc. The algae Dunaliella was used as a marine biosensor. This algae was easy to handle but not very sensitive against pentachlorophenol, but sensitive against atrazine. The experiments with Dunaliella were done with North Sea water. Escherichia coli and Pseudomonas putida were tested with spiked River Rhine water.

The biosensor is equipped with phototropic and heterotrophic organisms to detect a broader field of contaminants online in rivers and coastal waters. Thylakoids of Chenopodium rubrum have also been used to make the biosensor more specific for special herbicides and have a high specificity and sensitivity.

The Eucyanobacteria electrode operates with 8 parallel flow cells for each microorganism. The biosensor is equipped with a dynamic self calibrating alarm detection system. Actual measurements in the river matrix are continuously compared with those measurements from 10 minutes up to several hours in the past. If there is a change in the water quality, pollutants will be detected immediately. The system has been assessed for more than one year at the River Rhine at the German Dutch boarder in Bimmen and can be linked up to the WaBoLu-Aquatox monitoring system.The basis of this new methodology is the strong and highly specific binding of antigens (pesticides or their analogues) by antibodies, coupled with secondary electrochemical methods of signal transduction based on enzyme or whole cell reactions. An on-stream device, incorporating compound-specific catch systems (concentrator) in tandem with the signal transduction system, will be constructed. Rigorous monitoring of such a system with respect to the specificity of the concentrator; the effective pulsed release of analyte to the detector; and the sensitivity and specificity of electrochemical detection; by mass spectrometry, will allow full evaluation of the immuno-recognition and detection components. The goal is to demonstrate a broadly applicable pre-competitive prototype on-stream detector for a limited number of model and real pesticides, with a view to the generalised development of specific detectors to a wide range of environmentally significant compounds.